胰岛素样生长因子1受体在甲状腺相关性眼病眼眶脂肪细胞增殖和分化中的作用
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摘要
目的观察胰岛素样生长因子1(IGF-1)受体(IGF-1R)在甲状腺相关性眼病(TAO)眼眶脂肪组织中的表达及分布情况,探讨IGF-1对眼眶前脂肪细胞增殖和分化的影响。方法收集2014年9月至2015年1月期间入住第二军医大学长征医院的TAO组患者(n=6)和对照组患者(n=5)的眼眶脂肪组织,用蛋白质印迹法、实时定量PCR及免疫组化法检测两组患者眼眶脂肪组织中IGF-1R的表达及分布情况。分别提取两组患者的眼眶前脂肪细胞,用CCK-8法检测不同浓度(0、1、5、10、20nmol/L)IGF-1对细胞增殖能力的影响。体外分化细胞后用油红O染色法及蛋白质印迹法观察两组细胞的分化情况和IGF-1R的表达变化;实时定量PCR检测不同浓度(0、1、5、10、20nmol/L)IGF-1对TAO组细胞分化水平的影响。结果 TAO组患者眼眶脂肪组织中IGF-1R蛋白及mRNA的表达均高于对照组(P<0.01)。不同浓度IGF-1均可以促进眼眶前脂肪细胞的增殖,且在1~20nmol/L IGF-1作用下在TAO组细胞的增殖能力均高于对照组(P<0.05,P<0.01)。TAO组前脂肪细胞分化前、后细胞中IGF-1R的表达水平均明显高于对照组。相较于未加IGF-1组,1~20nmol/L IGF-1均促进TAO组前脂肪细胞中PPARγmRNA的表达(P<0.01);油红O染色检测结果显示随着IGF-1浓度的升高,脂滴合成逐渐增加(P<0.05,P<0.01)。结论 IGF-1R在TAO患者脂肪组织及前脂肪细胞中的表达较高,IGF-1可以促进TAO眼眶前脂肪细胞的增殖和分化。
Objective To observe the expression and distribution of insulin-like growth factor-1receptor(IGF-1R)in orbital adipose tissues of patients with thyroid-associated ophthalmopathy(TAO),and to explore the effect of insulin-like growth factor-1(IGF-1)on the proliferation and differentiation of orbital preadipocytes.Methods We harvested the orbital adipose tissues from 6 patients with TAO(TAO group)and 5controls(control group);the patients were treated in Changzheng Hospital,Second Military Medical University from Sep.2014 to Jan.2015.The expression and distribution of IGF-1Rin the orbital adipose tissues were examined by Western blotting analysis,real-time PCR and immunohistochemistry.The orbital preadipocytes were extracted in two groups,and the effects of different concentrations of IGF-1(0nmol/L,1nmol/L,5nmol/L,10nmol/L,and 20nmol/L)on cell proliferation were examined by CCK8.The differentiation of cells and the expression of IGF-1Rwere observed by oil red O staining and Western blotting;the effect of different concentrations of IGF-1(0nmol/L,1nmol/L,5nmol/L,10nmol/L,and 20nmol/L)on preadipocyte differentiation was detected by real-time PCR.Results The expressions of IGF-1Rprotein and mRNA in orbital adipose tissues in TAO group were significantly higher thanthose in the control group(P<0.01).The proliferation of orbital preadipocytes was promoted in both groups,and the cell proliferation in TAO group was significantly higher than that in control group when treated with 1-20nmol/L IGF-1(P<0.05,P<0.01).The expression of IGF-1Rin the TAO group was significantly higher than that in the control group before and after differentiation.Compared with IGF-1absent group,1-20 nmol/L IGF-1significantly promoted the expression of PPARγmRNA in orbital preadipocytes in TAO group(P<0.01).Oil red O staining results showed that the lipid droplet synthesis was significantly increased with the increase of IGF-1concentration(P<0.05,P<0.01).Conclusion IGF-1Ris highly expressed in the adipose tissue and preadipocytes in patients with TAO,and IGF-1can promote the proliferation and differentiation of preadipocytes in patients with TAO.
Objective To observe the expression and distribution of insulin-like growth factor-1receptor(IGF-1R)in orbital adipose tissues of patients with thyroid-associated ophthalmopathy(TAO),and to explore the effect of insulin-like growth factor-1(IGF-1)on the proliferation and differentiation of orbital preadipocytes.Methods We harvested the orbital adipose tissues from 6 patients with TAO(TAO group)and 5controls(control group);the patients were treated in Changzheng Hospital,Second Military Medical University from Sep.2014 to Jan.2015.The expression and distribution of IGF-1Rin the orbital adipose tissues were examined by Western blotting analysis,real-time PCR and immunohistochemistry.The orbital preadipocytes were extracted in two groups,and the effects of different concentrations of IGF-1(0nmol/L,1nmol/L,5nmol/L,10nmol/L,and 20nmol/L)on cell proliferation were examined by CCK8.The differentiation of cells and the expression of IGF-1Rwere observed by oil red O staining and Western blotting;the effect of different concentrations of IGF-1(0nmol/L,1nmol/L,5nmol/L,10nmol/L,and 20nmol/L)on preadipocyte differentiation was detected by real-time PCR.Results The expressions of IGF-1Rprotein and mRNA in orbital adipose tissues in TAO group were significantly higher thanthose in the control group(P<0.01).The proliferation of orbital preadipocytes was promoted in both groups,and the cell proliferation in TAO group was significantly higher than that in control group when treated with 1-20nmol/L IGF-1(P<0.05,P<0.01).The expression of IGF-1Rin the TAO group was significantly higher than that in the control group before and after differentiation.Compared with IGF-1absent group,1-20 nmol/L IGF-1significantly promoted the expression of PPARγmRNA in orbital preadipocytes in TAO group(P<0.01).Oil red O staining results showed that the lipid droplet synthesis was significantly increased with the increase of IGF-1concentration(P<0.05,P<0.01).Conclusion IGF-1Ris highly expressed in the adipose tissue and preadipocytes in patients with TAO,and IGF-1can promote the proliferation and differentiation of preadipocytes in patients with TAO.
引文
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[12]VALYASEVI R W,ERICKSON D Z,HARTENECK D A,DUTTON C M,HEUFILDER A E,JYONOUCHI S C,et al.Differentiation of human orbital preadipocyte fibroblasts induces expression of functional thyrotropin receptor[J].J Clin Endocrinol Metab,1999,84:2557-2562.
[13]JIA D,HEERSCHE J N.Insulin-like growth factor-1and-2 stimulate osteoprogenitor proliferation and differentiation and adipocyte formation in cell populations derived from adult rat bone[J].Bone,2000,27:785-794.
[14]ROOF S R,BOSLETT J,RUSSELLD,DEL RIO C,ALECUSAN J,ZWEIER J L,et al.Insulin-like growth factor 1 prevents diastolic and systolic dysfunction associated with cardiomyopathy and preserves adrenergic sensitivity[J].Acta Physiol(Oxf),2016,216:421-434.
[15]SATO T,NAGAFUKU M,SHIMIZU K,TAIRA T,IGARASHI Y,INOKUCHI J.Physiological levels of insulin and IGF-1 synergistically enhance the differentiation of mesenteric adipocytes[J].Cell Biol Int,2008,32:1397-1404.
[16]ZHAO P,DRNG Y,GU P,WANG Y,ZHOU H,HU Y,et al.Insulin-like growth factor 1promotes the proliferation and adipogenesis of orbital adipose-derived stromal cells in thyroid-associated ophthalmopathy[J].Exp Eye Res,2013,107:65-73.
[17]BURTURM D,ZHU Z,LU D,ANDERSON D M,PREWETT M,PEREIRA D S,et al.A fully human monoclonal antibody to the insulin-like growth factorⅠreceptor blocks ligand-dependent signaling and inhibits human tumor growth in vivo[J].Cancer Res,2003,63:8912-8921.
[18]MCCOY A N,KIM D S,GILLESPIE E F,ATKINS S J,SMITH T J,DOUGLAS R S.Rituximab(Rituxan)therapy for severe thyroid-associated ophthalmopathy diminishes IGF-1R+T cells[J/OL].J Clin Endocrinol Metab,2014,99:E1294-E1299.doi:10.1210/jc.2013-3207.
[2]FLYNN R W,MACDONALD T M,MORRIS A D,JUNG R T,LEESE G P.The thyroid epidemiology,audit,and research study:thyroid dysfunction in the general population[J].J Clin Endocrinol Metab,2004,89:3879-3884.
[3]KASPAR M,ARCHIBALD C,De B A,LI A W,YAADA M,CHANG C H,et al.Eye muscle antibodies and subtype of thyroid-associated ophthalmopathy[J].Thyroid,2002,12:187-191.
[4]VALYASEVI R W,ERICKSON D Z,HARTENECK D A,DUTTON C M,HEUFELDER A E,JYONOUCHI S C,et al.Differentiation of human orbital preadipocyte fibroblasts induces expression of functional thyrotropin receptor[J].J Clin Endocrino Metab,1999,84:2557-2562.
[5]WAKELKAMP I M,BAKKER O,BALDESCHI L,WIRESINGA W M,PRUMMEL M F.TSH-R expression and cytokine profile in orbital tissue of active vs inactive Graves’ophthalmopathy patients[J].Clin Endocrinol(Oxf),2003,58:280-287.
[6]TSUI S,NAIK V,HOA N,HWANG C J,AFIFIYAN N F,SINHA HIKIM A,et al.Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1receptors:a tale of two antigens implicated in Graves’disease[J].J Immunol,2008,181:4397-4405.
[7]VAREWIJCK A J,BOELEN A,LAMBERTS S W,FLIERS E,HOFLAND L J,WIERSINGA W M,et al.Circulating IgGs may modulate IGF-I receptor stimulating activity in a subset of patients with Graves’ophthalmopathy[J].J Clin Endocrinol Metab,2013,98:769-776.
[8]DOUGLAS R S,NAIK V,HWANG C J,AFIFIYAN N F,GIANOUKAKIS A G,SAND D,et al.B cells from patients with Graves’disease aberrantly express the IGF-1 receptor:implications for disease pathogenesis[J].J Immunol,2008,181:5768-5774.
[9]SALMIN W D,DAUGHADAY W H.A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro[J].J Lab Clin Med,1957,49:825-836.
[10]SHERLOCK M,TOOGOOD A A.Aging and the growth hormone/insulin like growth factor-Ⅰaxis[J].Pituitary,2007,10:189-203.
[11]SAX A T,JENKINSDG,DEVIN J L,HUGHES G I,BOLAM K A,SKINNER T L.The insulin-like growth factor axis:a biological mechanism linking physical activity to colorectal cancer survival[J].Cancer Epidemiol,2014,38:455-459.
[12]VALYASEVI R W,ERICKSON D Z,HARTENECK D A,DUTTON C M,HEUFILDER A E,JYONOUCHI S C,et al.Differentiation of human orbital preadipocyte fibroblasts induces expression of functional thyrotropin receptor[J].J Clin Endocrinol Metab,1999,84:2557-2562.
[13]JIA D,HEERSCHE J N.Insulin-like growth factor-1and-2 stimulate osteoprogenitor proliferation and differentiation and adipocyte formation in cell populations derived from adult rat bone[J].Bone,2000,27:785-794.
[14]ROOF S R,BOSLETT J,RUSSELLD,DEL RIO C,ALECUSAN J,ZWEIER J L,et al.Insulin-like growth factor 1 prevents diastolic and systolic dysfunction associated with cardiomyopathy and preserves adrenergic sensitivity[J].Acta Physiol(Oxf),2016,216:421-434.
[15]SATO T,NAGAFUKU M,SHIMIZU K,TAIRA T,IGARASHI Y,INOKUCHI J.Physiological levels of insulin and IGF-1 synergistically enhance the differentiation of mesenteric adipocytes[J].Cell Biol Int,2008,32:1397-1404.
[16]ZHAO P,DRNG Y,GU P,WANG Y,ZHOU H,HU Y,et al.Insulin-like growth factor 1promotes the proliferation and adipogenesis of orbital adipose-derived stromal cells in thyroid-associated ophthalmopathy[J].Exp Eye Res,2013,107:65-73.
[17]BURTURM D,ZHU Z,LU D,ANDERSON D M,PREWETT M,PEREIRA D S,et al.A fully human monoclonal antibody to the insulin-like growth factorⅠreceptor blocks ligand-dependent signaling and inhibits human tumor growth in vivo[J].Cancer Res,2003,63:8912-8921.
[18]MCCOY A N,KIM D S,GILLESPIE E F,ATKINS S J,SMITH T J,DOUGLAS R S.Rituximab(Rituxan)therapy for severe thyroid-associated ophthalmopathy diminishes IGF-1R+T cells[J/OL].J Clin Endocrinol Metab,2014,99:E1294-E1299.doi:10.1210/jc.2013-3207.